B-cell function in canine X-linked severe combined immunodeficiency

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gammac) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors and has a similar clinical phenotype to human XSCID. We have previously shown that the block in T-cell development is more profound in XSCID dogs than in genetically engineered gamma c-deficient mice. In this study we evaluated the B-cell function in XSCID dogs. In contrast to the marked decrease in peripheral B-cells in gamma c-deficient mice, XSCID dogs have increased proportions and numbers of peripheral B-cells as observed in XSCID boys. Canine XSCID B-cells do not proliferate following stimulation with the T-cell-dependent B-cell mitogen, pokeweed mitogen (PWM); however, they proliferate normally in response to the T-cell-independent B-cell mitogen, formalin-fixed, heat-killed Staphylococcus aureus. Canine XSCID B-cells are capable of producing IgM but are incapable of normal class-switching to IgG antibody production as demonstrated by in vitro stimulation with PWM and immunization with the T-cell-dependent antigen, bacteriophage PhiX174. Similar results have been reported for XSCID boys. Thus, it appears that gamma c-dependent cytokines have differing roles in human and canine B-cell development than in the mouse making the XSCID dog a valuable model for studying the role of these cytokines in B-cell development and function.     

Transfer of maternal cytokines to suckling piglets: in vivo and in vitro models with implications for immunomodulation of neonatal immunity

Maternal cytokines may play instructive roles in development of the neonatal immune system. However, cytokines in colostrum and milk and their transfer from mothers to neonates have not been well documented, except for TGF-beta. Swine provide a unique model to study lactogenic cytokines because the sow's impermeable placenta prohibits transplacental passage. We investigated IL-6 and TNF-alpha (pro-inflammatory), IFN-gamma and IL-12 (Th1), IL-10 and IL-4 (Th2) and TGF-beta1 (Th3) concentrations in sow serum and colostrum/milk and serum of their suckling and weaned piglets and in age-matched colostrum-deprived gnotobiotic piglets. All cytokines were detected in colostrum/milk and correlated with concentrations in sow serum except for mammary-derived TNF-alpha and TGF-beta1. Detection of IL-12 and TGF-beta1 in pre-suckling and colostrum-deprived gnotobiotic piglet serum suggests constitutive production: other cytokines were undetectable confirming absence of transplacental transfer. Peak median cytokine concentrations in suckling piglet serum occurred at post-partum days 1-2 (IL-4>IL-6>IFN-gamma>IL-10). The effects in vitro of physiologically relevant concentrations of the two predominant lactogenic cytokines (TGF-beta1 and IL-4) on porcine naive B cell responses to lipopolysaccharide (LPS) and rotavirus (RV) were investigated. High (10 ng/ml) TGF-beta1 suppressed immunoglobulin secreting cell responses to LPS and rotavirus; low concentrations (0.1 ng/ml) promoted isotype switching to IgA antibody. Interleukin-4 induced inverse dose-dependent (0.1 ng>10 ng/ml) isotype switching to IgA and enhanced IgM secreting cell responses to LPS and rotavirus. In summary, we documented the transfer and persistence of maternal cytokines from colostrum/milk to neonates and their potential role in Th-2 biased IgA responses and reduced immunologic responsiveness of neonates.     

Selective IgM deficiency and abnormal B-cell response in a foal.

Selective IgM deficiency was diagnosed in a 3-month-old Standardbred colt that was referred for chronic respiratory tract disease. Immunoglobulin quantification revealed normal IgG and IgA concentrations, but undetectable IgM concentration. Stimulation of blood lymphocytes with the T-cell mitogens concanavalin A and phytohemagglutinin yielded results within the normal range. However, stimulation with the B-cell mitogen lipopolysaccharide produced no response. A B-cell defect similar to that associated with several immunodeficiency disorders in people was suggested as the cause of the IgM deficiency in this colt.     

In vitro immunologic features of Weimaraner dogs with neutrophil abnormalities and recurrent infections

In vitro evaluation of cellular and humoral immunity in Weimaraner dogs with recurrent infections and abnormal neutrophil function revealed significantly lower serum immunoglobulin (Ig) G and M concentrations than control dogs. Lymphocyte blastogenesis in response to three different mitogens, interleukin-1 and -2 production, natural killer (NK) cell activity, and in vitro IgG and IgM production were similar to those of control dogs.     

Dynamic changes of immunoglobulin concentrations in pig colostrum and serum around parturition

The aim of the study was the determination of IgA, IgM and IgG concentrations in porcine serum and colostrum, in order to evaluate their variations in the perinatal period, as well as to clarify whether there is a correlation between colostrum intake, initial level of immunoglobulins (Ig) in piglet serum and development of their own immunity. The mean IgA, IgM and IgG concentrations in sow serum 10 days before parturition were 1.58, 6.12 and 39.56 mg/ml, respectively. Seven days later only the IgG level was insignificantly lower (34.94 mg/ml, p = 0.55), while concentrations of IgA and IgM increased to 2.25 and 7.25 mg/ml, respectively (p = 0.23 and 0.62, respectively). The mean initial IgG concentration in colostrum at farrowing was 118.5 mg/ml and differed between sows. The average value of IgA in colostrum at birth was 23.8 mg/ml and decreased to 7.85 mg/ml at 6 hours (h) and to 4.59 mg/ml at 24 h after the onset of farrowing. IgM concentration at birth was 12.1 mg/ml and decreased to 4.23 mg/ml at 24 h postpartum. Positive relationships were found between concentrations of IgM and IgA in serum of piglets at 14 and 56 days of life (r = 0.41 and 0.80, respectively, p < or = 0.05) as well as for IgG concentration in the piglets serum at 7 days and 56 days of age (r = 0.48, p < or = 0.05). The above observations suggest that there is a correlation between the level of Ig in piglet serum in the first days of life and improvement of their own immunity.     

Expression of tumor necrosis factor-alpha in IgM+ B-cells from bovine leukemia virus-infected lymphocytotic sheep

Tumor necrosis factor (TNF)-alpha is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-alpha expression in B-cells is limited. In this study, the expression of TNF-alpha in IgM(+) B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM(+) B-cells from three sheep with lymphocytosis constitutively transcribed TNF-alpha mRNA. Although TNF-alpha mRNA expression in IgM(+) B-cells was transiently up-regulated after cell culture, TNF-alpha mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membrane-bound TNF-alpha on IgM(+) B-cells was also augmented in lymphocytotic sheep. TNF-alpha expression in lymphocytotic sheep may support the proliferation of B-cells.     

Unusual Selective Immunoglobulin Deficiency in an Arabian Foal

A 10-month-old Arabian foal was evaluated for a suspected immunoglobulin (Ig) M deficiency. Decreased to nondetectable concentrations of IgM, IgA, and IgG (T), and a normal concentration of IgG, were present. Results of in vitro testing of the blood lymphocyte blastogenesis showed a weak response to the B-cell mitogen, lipopolysaccharide (LPS), but normal responses to T-cell mitogens. Results of postmortem examination showed synovitis of the left tibiotarsal and both scapulohumeral joints. Atrophy and edema of the lymph nodes and lymphocyte depletion in the thymus and spleen were seen. A subacute inflammatory infiltrate was observed in the kidney, synovium, liver, and brain. Etiologic agents were not identified. This case represents a previously unreported form of immunodeficiency disease in the horse.     

Development of ELISAs for the measurement of IgM and IgG subclasses in sera from llamas (Lama glama) and assessment of the humoral immune response against different antigens

Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.     

The absorption of colostral immunoglobulins in newborn piglets. III. Influence of the duration of colostrum administration

The gastrointestinal tract of newborn piglets is permeable for intact immunoglobulins ingested with the colostrum. The duration of this passage was investigated by administering hourly rations of 25 ml of either porcine or bovine colostrum for 6, 12, 18 or 24 hrs after birth. The plasma concentrations of the subclasses porcine IgG, IgM and IgA or bovine IgG1, IgG2, IgM and IgA were determined at 12, 18 and 24 hrs after birth and on days 3 and 6. Feeding periods of 6 hrs resulted in plasma Ig levels of the same order of magnitude as observed in natural rearing. These levels were not substantially increased after prolonged feeding. The 6% gain from 6 to 12 feedings seen with porcine colostrum as compared with a gain of 24% for bovine colostrum points at an earlier closure of the intestinal wall for the species-specific proteins. There was no further increase of Ig permeation after 12 hourly feedings. Growth performances and losses were identical in all groups.     

Immune defects in simian acquired immunodeficiency syndrome

We recently reported a Simian Acquired Immunodeficiency Syndrome (SAIDS) in rhesus macaques at the California Primate Research Center. Here, we studied in vitro lymphocyte response to the mitogens Concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) with and without interleukin 2 (IL-2). Immunoglobulin (IgG and IgM) and complement (C3 and C4) concentrations were determined by radial immunodiffusion. T helper and T suppressor lymphocytes were identified with the monoclonal antibodies OKT4 and OKT8. Concentrations of IgG and IgM were significantly (p less than .05) decreased. Complement component C3 did not change but C4 was increased. The absolute lymphocyte count decreased but the OKT4:OKT8 ratio was unchanged from controls. A decreased lymphocyte response to all mitogens occurred early and became more severely depressed near death. IL-2 caused a complete or partial restoration of the response to the mitogens CON A and PHA. Both the humoral and cell mediated immune responses are affected in SAIDS. The role of IL-2 in this immune defect must be studied further.     

Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae)

Antigen-specific antibody responses, T cell subsets and cytokine profiles were studied in 7 heifers naturally infested by Hypoderma sp. in Northwestern Spain. Immunoglobulin G (IgG) levels increased significantly at the end of the endogenous life cycle of the parasite (Mr). Similarly, IgG1 subclass increased considerably when first instars (L1) started their migration towards the back (Nv-Ja), whereas IgG2 increased earlier, coinciding with the arrival of L1 to the resting sites (Jn-Jl). Both subclasses decreased significantly when L3 began to leave the host. IgM levels and CD4 and CD8 profiles hardly oscillated throughout the life cycle of the parasite into the host. The CD4/CD8 ratio showed helper T cell predominance. Serum interferon-γ (IFN-γ) concentrations decreased from October to the end of the study. Interleukin 4 (IL-4) concentrations decreased in January and increased in February and May. There were a significant positive relationship between IL-4 and IgG2 subclass and a negative correlation between IFN-γ, IgG and IgG1 and also between IgM and CD2 and CD8 counts. These results suggest that in the early phases of natural primoinfestations by Hypoderma there is a slight predominance of a Th1 response, characterized by high IgG2 and IFN-γ levels, which is followed by a Th2 response with a clear predominance of IgG1 and IL-4.     

Colostral volume and immunoglobulin G and M determinations in mares

Colostral volume and IgG and IgM concentrations were determined in 6 multiparous mares at foaling and them every 2 hours from 16 to 20 hours after parturition. Serum IgG and IgM concentrations at foaling also were determined in each mare. The rate of mammary secretion was 292 +/- 26 ml/h (range, 202 to 389 ml/h), and the colostral volume was 5.1 +/- 0.5 L (range, 3.2 to 7.0 L). The colostral IgG and IgM contents were 440 +/- 106 g (range, 199 to 855 g) and 3.1 +/- 0.9 g (range, 0.7 g to 7.1 g), respectively. There was no significant correlation between serum and initial colostral IgG and IgM concentration or between serum and total colostral IgG or IgM values. The colostral IgG and IgM concentrations at foaling correlated well with the total colostral IgG and IgM contents, respectively. The initial 250 ml of colostrum contained 10 +/- 1.4% (range, 6.0 to 13.9%) and 6 +/- 1.0% (range, 2.4 to 8.5%) of the total IgG and IgM contents, respectively, and the initial 500 ml of colostrum contained 20 +/- 2.7% (range 12.0 to 27.1%) and 14 +/- 1.2% (8.2 to 17%) of the total colostral IgG and IgM contents, respectively.     

Large-scale production of porcine mature interleukin-18 (IL-18) in silkworms using a hybrid baculovirus expression system

In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 microg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-gamma (IFN-gamma) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future.     

Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2)

Immunological impairment by porcine circovirus type 2 (PCV2) infection is well documented in pigs suffering from postweaning multisystemic wasting syndrome. Nonetheless, little is known about immune status of pigs that remain PCV2 subclinically infected. Thus, seven pigs successfully infected in an experimental inoculation and without developing disease and nine control non-inoculated pigs were examined. Serological, virological and immunological determinations were done throughout ten weeks post-infection (PI). At week 3 PI, inoculated animals presented the peak of viremia and produced higher levels of IL-10 than the controls; correlation between viral load and IL-10 amounts was observed (p<0.05). Also, the ratio IgM/IgG suffered a shift skewing IgM production towards an IgG response. By 10 weeks PI, levels of IL-10 disappeared and the viremia decreased. In summary, subclinically PCV2-infected pigs developed a transient PCV2-specific IL-10 response during the viremic phase of infection which coincided with the inversion of the IgM/IgG ratio.     

Immunoglobulins and lesions in aborted bovine foetuses

IgG and IgM in serum from 164 aborted and 59 abattoir foetuses were measured by radial immuno-diffusion. Ig was detected in 133 (81.1%) aborted and 6 (10.2%) abattoir foetuses. The difference was statistically significant (P<0.01).

More than 20 mg/100 ml IgG was found in 90 (54.9%) aborted foetuses and 1 (1.7%) abattoir foetus. More than 20mg/100 ml IgM was present in 62 (37.8%) aborted foetuses and 2 (3.4%) abattoir foetuses. In each case the difference was statistically significant (P<0.01).

The mean concentration of IgG was 128 mg/100 ml in the aborted and 13 mg/100 ml in abattoir foetuses. The mean concentration of IgM was 34 mg/100 ml in aborted foetuses and 1.4 mg/100 ml in abattoir foetuses.

Tissues from 143 aborted foetuses were examined histologically. Serum from 86 of the 143 had more than 20 mg/100 ml IgG, IgM or both; of these, 79 (91.9%) had well defined or possible lesions. No lesions were detected in 7 (8.1%).

Serum from 32 of the 143 foetuses had 1 to 20 mg/100 ml IgG, IgM or both. Well-defined or possible lesions were seen in 24 (75.0%) of these. No lesions were detected in 8 (25.0%).

Serum from 25 of the 143 foetuses had no detectable Ig. Of these, 11 (44.0%) had well defined or possible lesions. No lesions were seen in 14 (56.0%).

If results of further investigation indicate the foetus does not frequently produce excess Ig in response to stimuli other than intrauterine infection, then measuring foetal Ig by RID may prove valuable as a first step in diagnosing infectious bovine abortion.     

Genetics of immunocompetent traits in a synthetic broiler dam line

Three hundred and three chicks of both sexes, from a synthetic dam line (SDL) of broiler chickens, were studied for economic traits (body weights at 4, 5 and 6 weeks of age) and immunological traits (humoral and cell mediated immune responses, and serum lysozyme concentration). The objective was to evaluate these traits and to estimate their genetic and non-genetic parameters. The humoral immune response was assessed by estimating the antibody response to sheep red blood cells using the haemagglutination (HA) test and serum IgG concentration using single radial immunodiffusion (SRID). The cell mediated immune (CMI) response was estimated as in vivo response to a mitogen (PHA-P). Serum lysozyme was measured by lysoplate assay. Least squares means for body weight at 4, 5 and 6 weeks were 684 +/- 20, 920 +/- 19 and 1205 +/- 28 g, HA titre was 6.289 +/- 0.246, CMI was 0.438 +/- 0.015 mm, lysozyme was 1.860 +/- 0.047 microg/ml and IgG was 6.287 +/- 0.194 mg/ml. There was an effect of sire on HA titre and on body weight at 4, 5 and 6 weeks of age; males were heavier than females. Heritability estimates were high for body weights but low for immunological traits. Phenotypic correlations (rp) among body weights were high and positive but were very low between body weights and most immunological traits. Among the immunological traits all rp were very low. Genetic correlations (rg) of body weights were positive and medium to high with CMI and HA and negative with serum IgG.     

An immunologic investigation of canine eosinophilic bronchopneumopathy

Immunologic variables in dogs with eosinophilic bronchopneumopathy (EBP) have not been extensively evaluated. The aim of this study was to determine immunoglobulin (Ig) concentrations and to perform phenotypic subtyping of lymphocytes in the bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of 12 dogs with EBP at the time of diagnosis (TD) and to compare these data with those obtained in healthy dogs, as well as in EBP dogs after antibiotic therapy (TAB) and during corticosteroid treatment (TM). Matched samples of serum and BALF were used to determine Ig concentrations (IgG, IgM, and IgA) by capture enzyme-linked immunosorbent assay (ELISA), from which a secretory index (SI) was calculated. Lymphocyte subpopulations were studied in the BALF and PB by flow cytometry. Log values of BALF IgM and IgA were significantly higher (0.64+/-0.05 and 1.06+/-0.13, respectively) in EBP dogs at TD than in controls and then tended to decrease at TM (0.55+/-0.03 and 1.02+/-0.17, respectively). A calculated SI for IgA was not significantly increased. In the BALF of dogs with EBg the CD4: CD8 was significantly (P < .05) higher (22.6+/-30.3) than in controls (3.2+/-1.9), due to significantly higher CD4+ T cells and lower CD8+ T cells. At TM, the BALF T-cell percentages returned to normal (2.4+/-0.6). We propose that the influx of eosinophils into the airway of dogs with EBP is at least in part mediated by cytokines derived from CD4+ T cells. Further studies of canine cytokines and chemokines will help determine whether canine EBP involves type I hypersensitivity mechanisms regulated by Th2 lymphocytes.     

Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

Interleukin-6 expression in gut of parasite challenged sheep

Following challenge with Trichosirongylus colubrifonizis, increased numbers of T-cells and immunoglobulin responses are seen in the intestine of sheep immunised by repeated infection with live worms. IL-6 mRNA expression in the small intestine from T. colubriformis-immunised and naive sheep was determined by in situ hybridisation, whereas CD4(+), IgA(+), IgG(+) cells in the gut were evaluated by immunohistochemistry. There was constitutive expression of IL-6 mRNA by cells in the naive gut, and the number of these cells was increased by parasite challenge. There were corresponding increases in numbers of CD4(+) and TCR gamma/delta(+) T-cells and IgG(+) B-cells. Our data are consistent with a role for IL-6, perhaps produced by CD4(+) and/or TCR gamma/delta(+) T-cells or B-cells, in B-cell terminal differentiation. Infiltration of B-cells, particularly IgG(+) B-cells, may reflect parasite immunity in the host.     

In vitro system for immunoglobulin class switching using BHK cells transfected with murine recombinant CD40 ligand.

To develop an in vitro system for mouse immunoglobulin (Ig) class switching, the expression vector of murine CD40 ligand (CD40L) which is expressed on T cells was transfected to BHK cells. By using culture plates coated with the BHK cells expressing the recombinant CD40L, Ig class switching of splenic B cells was examined. The CD40L mRNA was cloned from splenic T cells of BALB/mice activated with anti-CD3 antibody in vitro. As the No.593 base in the open reading frame sequence of the CD40L from BALB/c spleen differed from T to G, when compared with the known sequence from C57BL/6, one of the BALB/c-derived clones was reconstructed to the known CD40L by site-directed mutagenesis. Splenic B cells from BALB/c were induced secretion of Ig isotypes, IgM, IgG1 and IgE when cultured on two types of BHK cells, the transfected BHK cells with a CD40L clone from BALB/c and the transfected BHK cells with the reconstructed CD40L clone, in the presence of IL-4. However, when splenic B cells from C57BL/6 were cultured on the same systems, the B cells produced Ig isotypes, IgM, IgG1, IgG2a, IgG2b, IgG3 and IgE. In the similar experiments using the transfected BHK cells with a empty vector and the normal BHK cells, none of B cells produced any Ig isotypes other than IgM. These results indicate that Ig class switching of murine B cells can be induced by using these two types of CD40L-expressing BHK cells in vitro.